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Factor V Leiden Detection and Genotyping

Factor V Leiden Detection and Genotyping . August 6, 2008 Marylin Bicak. Objective . 1. To provide an overview of the Factor V Leiden assay 2. To explain the pathophysiology and epidemiology of the Factor V Leiden mutation. 3. To evaluate the efficacy of the assay. Pathophysiology.

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Factor V Leiden Detection and Genotyping

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  1. Factor V Leiden Detection and Genotyping August 6, 2008 Marylin Bicak

  2. Objective • 1. To provide an overview of the Factor V Leiden assay • 2. To explain the pathophysiology and epidemiology of the Factor V Leiden mutation. • 3. To evaluate the efficacy of the assay.

  3. Pathophysiology • inherited condition • autosomal dominant • chromosome 1, gene F5 • Single point mutation, G1691A (arginine to glutamine at 506th amino acid • mutation prevents inactivation of factor V in clotting process (resistant to inactivation by Protein C) • overproduction of thrombin= excess fibrin formation • excess clotting (DVT and PE)

  4. Epidemiology • Most common inherited coagulation disorder in U.S. • 5% Caucasians • 2% Hispanics • 1.2% African Americans • <0.5% Asian Americans • Heterozygous= 3-8 fold increase risk of thrombosis • Homozygous= 30-140 increase risk of thrombosis • risk factors • treatment

  5. Factor V Leiden Assay • 1. Extraction- MagNA Pure Compact Nucleic Acid Isolation Kit and instrument (Roche Diagnostics) • 2. Amplification/Detection/Genotyping- Factor V Leiden Kit and LightCycler 2.0 instrument (Roche Diagnostics)

  6. Extraction • Specimen- EDTA whole blood (200 ul whole blood= 100 ul purified product) • MagNA Pure Kit- pre-sealed reagent cartridge; disposable pipette tip tray assembly; sample tube; elution tube • MagNA Pure Compact Instrument-automated method based on magnetic glass particle technology

  7. Principle of Magnetic Glass Particle Technology • Four step process: 1-lysis; 2-bind to magnetic particles; 3-wash; 4-elution

  8. Nucleic Acid Isolation

  9. Amplification-real-time PCR • target- 222 bp fragment of Factor V gene • Factor V Leiden kit- master mix reagents (primers and probes) • LightCycler 2.0 instrument- rapid- 45 cycles (30 min) • Cycle temperatures- denaturation 95C; annealing 60C; elongation 72C • thermal cycler- heat/ambient air cycle • reaction vessel- 20 ul glass capillary

  10. Detection • FRET (fluorescence resonance energy transfer)

  11. Genotyping • Melting Curve Analysis

  12. Summary • Limitations- technical process/ physical space/ instrumentation • Erroneous results- a. false positive (3 rare mutations span same mutation probe) and b. patient sample with elevated WBC • Overall, innovative system and important diagnostic tool for clinical lab

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